一株同时携带质粒介导KPC-2、DHA-1基因对碳青霉烯类药物耐药的产气肠杆菌  

A clinical isolate of carbapenem-resistant Enterobacter aerogen with plasmid-mediated KPC-2 and DHA-1 genes

作　　者：蒯守刚[1] 王卫萍[2] 裴豪[1] 范明[1] 刘君[2] 周希科[2] 尚忠波[2] 邵海枫[1] 

机构地区：[1]无锡市第五人民医院,江苏无锡214005 [2]南京军区南京总医院,江苏南京210002

出　　处：《检验医学》2014年第4期375-379,共5页Laboratory Medicine

摘　　要：目的研究一株临床分离的碳青霉烯类药物耐药产气肠杆菌的耐药机制和耐药基因传播机制。方法采用琼脂稀释法检测菌株对抗菌药物的最低抑菌浓度(MIC),采用质粒接合试验、质粒提取、DNA分子杂交、等电聚焦电泳(IEF)、聚合酶链反应(PCR)、DNA测序和外膜蛋白分析研究菌株的耐药基因及其传递机制。结果 IEF显示临床分离的产气肠杆菌株含有3条β-内酰胺酶条带,等电点(pI)分别为5.4、6.7和7.8。PCR扩增及测序结果表明它们分别为TEM-1(pI 5.4)、KPC-2(pI 6.7)、DHA-1(pI 7.8)β-内酰胺酶。接合菌含有2条β-内酰胺酶条带,pI为6.7和7.8。接合试验、质粒提取和分子杂交试验结果显示KPC-2和DHA-1基因定位于同一个约56 kb大小的质粒上。与临床野生产气肠杆菌株相比,外膜蛋白电泳分析显示耐药分离株缺失41 000外膜蛋白。结论产气肠杆菌分离株对碳青霉烯类抗菌药物耐药可能由A类2f组KPC-2酶介导,DHA-1酶合并外膜蛋白缺失也可能与碳青霉烯类药物耐药机制形成有关。Objective To investigate the resistance and transmission mechanisms of a clinical isolate of carbapenem-resistant Enterobacter aerogen.Methods The minimal inhibition concentrations （MIC）of antimicrobial agents were determined by agar dilution method,and plasmid conjugation experiment,plasmid extraction and DNA molecular hybridization, isoelectric focusing electrophoresis （IEF ）, polymerase chain reaction （PCR ）, DNA sequencing and outer-membrane protein analysis were used for analyzing the resistant gene and transmission mechanism. Results IEF showed that there were 3 bands of beta-lactamases with isoelectric point （pI）of 5.4,6.7 and 7.8 in the clinical isolate of carbapenem-resistant Enterobacter aerogen.These 3 bands of beta-lactamases were confirmed to be TEM-1 （pI 5.4）,KPC-2（pI 6.7）and DHA-1 （pI 7.8）by PCR amplification and DNA sequencing,and 2 of them （pIs of 6.7 and 7.8）were found that they can be transferred by plasmid conjugantion experiment.KPC-2 and DHA-1 genes were located on an about 56 kb plasmid by plasmid conjugation experiment,plasmid extraction and DNA molecular hybridization.Outer-membrane protein electrophoresis analysis revealed that a 41 000 outer-membrane protein was absent in the clinical isolate of carbapenem-resistant Enterobacter aerogen comparing with clinical wild-type Enterobacter aerogen.Conclusions The clinical isolate of Enterobacter aerogen resistant to carbapenem produces a plasmid-mediaed carbapemase KPC-2 which belongs to Group 2f,Class A beta-lactamase.DHA-1 enzyme with outer-membrane protein absence may be related with the resistant mechanism of carbapenem-resistance in the isolate of Enterobacter aerogen.

关 键 词：产气肠杆菌 碳青霉烯类 质粒 Β-内酰胺酶 肠杆菌科 

分 类 号：R378.2[医药卫生—病原生物学]